This invention relates to medicine and, more particularly, to contact-free clinical diagnostics of proliferation areas in biological tissues and their localisation areas in vivo in a live organism on the basis of endogenous porphyrin fluorescence.
Both the known and the proposed proliferation area diagnostics methods are based on the ability of porphyrins to be localised selectively in proliferating tissues (The Big Medical Encyclopaedia, RU, Moscow, Sovetskaya Entsiklopedia Publishers, 1983, Volume 20, Page 349)
The known proliferation area diagnostics methods in oncology are the introduction of exogenous porphyrins to a patient (Photodynamic Therapy and Fluorescent Diagnostics of Malignant Tumours with the Use of Photogem Preparation, V.I. Chissov et al., Khirurgiya (Surgery), No. 12, 1994, p. 3-6; Clinical Fluorescent Tumour Diagnostics with Photosensitinogen Photogem, V.I. Chisov et al. Khirurgia (Surgery), No. 5, 1995, p. 37-41; (see also references in those articles) or the introduction of the preparations that stimulate active generation of endogenous porphyrins in a patient""s organism (Pharmacokinetic of Endogenous Porphyrins Induced by 5-Aminolevulinic Acid as Observed by Means of Laser Induced Fluorescence from Several Organs of Tumour-Bearing Mice, Ronald Sroka, Reinhold Baugartner, Wolfgang Beyer, Liebwin Gossner, Tarek Sassy, Susanne Stocker., BIOS""95, 4-10 February. 1995, SPIE Proc. Vol. 2387, pp. 22-29) and, after some time, sufficient for selective re-distribution of the introduced exogenous porphyrins in tissues or stimulation of the generation and re-distribution of endogenous porphyrins, consecutive irradiation of small segments of the surface of the tissue under examination with the wave length which falls into the porphyrin fluorescence excitation band, with the recording of the fluorescence band simultaneously to that.
The main disadvantage of these diagnostics methods is their invasiveness, that is, the need for the introduction to a patient of either exogenous porphyrins or the substances which stimulate active generation of endogenous porphyrins in the organism. Increase in the content of porphyrins in the organism results in all negative developments typical of porphyria, such as, porphyrin exchange disfunction, including significant increase in photosensitivity of the organism. In this connection the given methods cannot be used in primary diagnostic examination, particularly in case of public preventive monitoring of the population.
The disadvantages of the indicated diagnostics methods also include their low performance caused first of all by quite a long period of time required for the selective re-distribution of the introduced exogenous porphyrins in tissues or the stimulation of the generation and re-distribution of endogenous porphyrins. Besides, the indicated methods record fluorescence bands which implies consecutive analysis of the tissue under examination from one point to another. Apart from the optic properties of the tissue proper, the size of a point, that is the area of tissue being examined at a given time, is also determined by the apertures of the radiation which induces fluorescence and the optical fibres which receive the fluorescent response and the location of their front-sides in relation to the tissue under examination. This results in low spatial resolution and poor reproducibility of the measurement results for the given methods. If it is necessary to examine big areas of various organs, skin etc., the probability of xe2x80x9cblanksxe2x80x9d, that is, the segments of the tissue under diagnostics that escape examination, is high. Besides, a disadvantage of the given methods is that it is difficult to document the location of proliferation areas and their localisation boundaries.
A known cancer identification method (Tumor detection in HpD-sensitized mice with fluorescence lifetime imaging, R. Cubeddu, G. Canti, A. Pifferi, P. Taroni, and G. Valentini, SPIE Proc. Vol, 2972, pp. 148-153) is the introduction of an exogenous derivative of hematoporphyrin and after some time, sufficient for its selective re-distribution in tissues, the exposure of the tissue under examination to short radiation pulses which induce fluorescence of the hematoporphyrin derivative with the wave length of 405 nm and the recording of the fluorescent image with time delay in relation to the generating radiation pulse so as to identify only the fluorescent response of the substance to be identified.
The disadvantages of this method include its invasiveness caused by the need for introducing exogenous fluorophore, as well as the complexity, high cost and relatively low resolution of the equipment required for producing the image with a millimicrosecond time delay in relation to the pulse of the radiation which induces fluorescence.
A known method of the diagnostics of affected tissues (Mechanisms of ratio fluorescence imaging of diseased tissue, Jianan Qu, Calum MacAulay, Stephen Lam and Branko Palcic, SPIE Proc. Vol. 2387, pp. 71-79), is the irradiation of the tissue segment under examination inducing endogenous fluorophores fluorescence with the wave length of 442 nm and the recording of two fluorescent images of the same tissue segment at the wave lengths of 500 nm and 630 nm. Then the ratio between two fluorescent images produced in the red and in the green wave length bands is taken, and the degree of effect on the tissue is determined by this ratio, if it exceeds a certain value.
The disadvantage of this method is relatively low sensitivity which dictates the need for using expensive cameras which brightness amplifiers. This is caused first of all by the fact that the blue radiation (442 nm) penetrates the tissue to quite insignificant depth and, respectively, can induce the fluorescence of only the fluorophores located close to the surface. Thus, it is difficult to carry out the diagnostics of defects under the surface. The optical properties of biological tissues in the blue (442 nm), green (500 nm) and red (630 nm) spectral bands are different to a significant extent and can vary from one patient to another which results in the need for using special algorithms for processing diagnostic information. Besides, railcar with the wave length of 442 nm falls into the band which induces the fluorescence of a whole range of endogenous fluorophores, such as collagen, elastin, porphyrins and their complexes with proteins, etc. Also, the concentration of porphyrins and their fluorescing complexes with proteins is frequently significantly lower than the concentration of other fluorophores. The fluorescence bands of various endogenous fluorophores are quite broad and partially overlap, therefore it is difficult to differentiate between them in case of their simultaneous excitation. Fluorescence of the fluorophores the concentration and distribution of which in tissues do not provide the required information regarding tissue condition is a confusing noise factor which distorts the informative signal.
A known method of detecting skin anomalies (Method of detecting anomalies of the skin, more particularly melanomae, and apparatus for carrying out the method, Gerhard Martens, Erhard P. H. Gunzel, U.S. Pat. No. 5,363,854, Nov. 15, 1994) is as follows: a skin segment under examination is irradiated in the ultraviolet spectrum band; the fluorescent image is recorded, and then the same skin segment is exposed to visible light, and the reference image of the same skin segment is recorded as seen in the visible band. Then a third image is produced where the brightness of each point is equal to the ratio between the brightness values of the first two images in the corresponding points. Skin segment anomalies are determined by brightness distribution in the third image.
The disadvantage of this method is its low sensitivity caused by the fact that wide band ultraviolet radiation induces the fluorescence of virtually all the fluorophores existing in the tissue under examination. It is possible to single out the fluorescent signal of one type of fluorophores which contains diagnostic information is possible only if the concentration of the fluorophores in question significantly exceeds that of other fluorophores. In a general case, this in turn is possible only in case of artificial invasive increase in their concentration. Apart from that, extremely low depth of penetration of ultraviolet radiation into skin tissue can be noted.
The proposed invention is aimed at increasing the precision, reliability and sensitivity of the diagnostics of proliferation areas in tissues in vivo, increasing the speed of diagnostics and eliminating the need for invasive intervention into the patient""s organism.
The indicated technical tasks are addressed as follows:
the tissue segment is evenly exposed to monochromatic radiation within the wave length band of 630 to 645 nm during the first period, and the fluorescent image of the tissue segment under examination is recorded in the spectral wave length band of 650 to 730 nm; the choice of the duration of the exposure and, respectively, the recording of the fluorescent image is based on the fluorescent signal intensity level and the dynamic range of the recording device; if the fluorescent signal intensity level equals the photon noise level or the internal noise of the recording device, recording is carried out in several cycles where the duration of each of them is determined with regard to the dynamic range value of the recording device, and the number of the cycles and, respectively, the total duration of the recording is determined on the basis of the required degree of statistical averaging of noise. The resulting fluorescent image is produced by way of averaging the brightness values of the corresponding points of the image for all recording cycles, determining the significant range of the brightness values of the averaged image and broadening this range by way of recalculation with regard to the entire dynamic range of the information display device.
The tissue segment under examination is evenly exposed to white light during the second period, and its reference colour image is recorded with the same angle and scale as in the case of recording the fluorescent image.
The areas where proliferation intensity changes in the tissue segments under examination are determined by formal signs in the fluorescent image, and the localisation areas are determined by comparing the fluorescent image with the reference colour image with the use of the coordinate grid, reference points applied thereon or by way of overlaying the images.
Besides, two auxiliary fluorescent images are recorded additionally during a diagnostic session with the use of the same equipment, in the same spectral band, with the same scale, wave length, density, power and duration of the exposure of the radiation inducing fluorescence as in the case of recording the fluorescent image of the tissue under examination.
The first auxiliary fluorescent image, the fluorescent image of the test object which is, for example, a cassette with several compartments filled with a stable fluorophore solution with known concentrations which differ by a known number of times from one compartment to another; the fluorophore solution should have the spectral bands of excitation and fluorescence which are similar to those of the endoporphyrins to be identified and their fluorescing protein complexes in the tissues under examination, and have the absorption and scattering values in the spectral bands under consideration which are similar to the corresponding values of the tissues under examination.
The first auxiliary fluorescent image is used to monitor (check) the diagnostic process sensitivity for ensuring its authenticity throughout the service life of the diagnostic equipment. Besides, by comparing the brightness values of particular segments in the fluorescent image of the tissue under examination and the first auxiliary fluorescent image (with the test object), the concentration of endogenous porphyrins and their fluorescing complexes with proteins in the tissue under examination is estimated.
The second auxiliary fluorescent image is the fluorescent image of the natural proliferation area of the same patient (for example, the growth areas of an unaffected nail plate).
The second auxiliary fluorescent image is used to determine contrast between the segments which correspond to actively proliferating tissue and are adjacent to poorly proliferating or non-proliferating tissue, as well as the brightness gradient between them. A similar procedure is applied to the fluorescent image of the tissue under examination, and the comparison is carried out between the contrast and brightness gradient at the second auxiliary fluorescent image. The degree of proliferation of the tissue under examination is estimated on the basis of the comparison results. The brightness values which are averaged for the recording period, as well as the brightness values averaged by the space which corresponds to the tissue segments in question are used for establishing contrast and brightness gradient in the fluorescent images.
Two more auxiliary monochrome images of the tissue under examination are recorded additionally with the same angle and scale as in the case of recording the fluorescent image to study the tissues that contain segments with (significantly) different absorption and scattering values in the spectrum bands used: one image (or the third auxiliary image) is recorded at the wave length of the used source of the radiation inducing fluorescence with even exposure of the tissue under examination to radiation from this source; another image (or the fourth auxiliary image) is recorded in the same spectral band where the fluorescent image is recorded, but with even exposure of the tissue under examination to radiation from an additional source in the same spectral band (in the fluorescence band used).
A coordinate grid and reference points are also applied to the third and fourth monochrome auxiliary images, or provisions are made for the possibility of overlaying or combining with the fluorescent and colour reference images of the tissue under examination.
The third and fourth monochrome auxiliary images are used to assess optical absorption and scattering values of the tissue segment under examination in the spectral bands that correspond to the selected bands of excitation and fluorescence of endogenous porphyrins and their fluorescing complexes with proteins. The location of local changes in optical absorption and scattering values is determined by comparing the third and fourth monochrome auxiliary images with the fluorescent and colour reference images of tissue under examination with the use of the coordinate grid or reference points applied thereon or by overlaying them on each other.
In order to implement the claimed method, a proliferation area diagnostics device is proposed; the layout of the device is displayed at FIG. 1; the device comprises a monochrome source of the radiation inducing the fluorescence of endogenous porphyrins and their complexes with proteins 3, fluorescence image recording unit 13, reference image recording unit 10, a computer with graphic data printing, documenting and storage devices 14, 15, 16. The distinctive feature of this device is that the monochrome source of the railcar inducing the fluorescence of endogenous porphyrins and their complexes with proteins operates within the wave length band of 630-645 nm, the fluorescent image recording unit is implemented in the form of a monochrome CCD-camera with variable frame exposure time and comprises additionally a white light source for exposing the tissue surface under examination when recording the reference colour image 1, a source of monochrome radiation within the wave length band of 650 to 730 nm for exposing the tissue under examination when recording the fourth monochrome auxiliary image 2, a source of lighting the laboratory premises in the visible spectrum band which does not radiate in the wave length band of above 650 nm 4, a radiation switching unit for sources 1, 2, 3 and a unit for making a collinear ray configuration 5, a unit of the collinear illumination of the tissue under examination from sources 1, 2, 3 and receiving reflected signals and the fluorescent signal 6, image splitting unit 11, radiation filtration unit 12, a processor for video signals, synchronisation and control signals 9 linked to a computer, the fluorescent image recording unit, the reference image recording unit, the unit for switching radiation sources and making a collinear ray configuration, the radiation filtration unit and radiation sources 1, 2, 3, 4.
The proposed device operates as follows
Radiation coming from source 3 through switching unit 5 and collinear illumination and receiving unit 6 evenly illuminates the tissue segment of object 7 under examination. The fluorescent response from the tissue segment of object 7 under examination is processed through collinear illumination and receiving unit 6 by lens 8 to be made an image at the receiving element of recording unit 13 through image splitting unit 11 and radiation filtration unit 12. The recording mode is input by the processor for video, synchronisation and control signals 9. The video signal comes from recording unit 13 to processor 9 which processes the recorded fluorescent image and transmits it to computer 14 and further to data printing and storage devices 15 and 16.
During the next period radiation coming from white light source 1 through switching unit 5 and collinear lighting and receiving unit 6 evenly illuminates the tissue segment of object 7 under examination. The light reflected from the tissue segment of object 7 under examination is processed through collinear illumination and receiving unit 6 by lens 8 to be made an image at the receiving element of recording unit 10 through image splitting unit 11. The recording mode is input by video, synchronisation and control signal processor 9. The video signal comes from recording unit 10 to processor 9 where the recorded reference image in colour is processed, if necessary, and is transmitted to computer 14 and further to data printing and storage devices 15 and 16.
The third auxiliary monochrome image is recorded with even illumination of the tissue segment of object 7 under examination by radiation from source 3 through switching unit 5 and collinear illumination and receiving unit 6. The light reflected from the tissue segment of object 7 under examination is processed through collinear illumination and receiving unit 6 by lens 8 to be made an image at the receiving element of recording unit 10 through image splitting unit 11 (or at recording unit 13 through image splitting unit 11 and radiation filtration unit 12; the filter is changed in this case upon a signal from processor 9). The recording mode is input by video, synchronisation and control signal processor 9. The video signal goes from recording unit 10 (or 13) to processor 9 where the recorded auxiliary image is processed, if necessary, and transmitted to computer 14 and further to data printing and storage devices 15 and 16.
The fourth auxiliary monochrome image is recorded with even illumination of the tissue segment of object 7 by radiation from source 2, through switching unit 5 and collinear illumination and receiving unit 6. The light reflected from the tissue segment of object 7 under examination is processed through collinear illumination and receiving unit 6 by lens 8 to be made an image at the receiving element of recording unit 10 through image splitting unit 11 (or at recording unit 13 through image splitting unit 11 and radiation filtration unit 12; the filter is changed in this case upon a signal from processor 9). The recording mode is input by video, synchronisation and control signal processor 9. The video signal goes from recording unit 10 (or 13) to processor 9 where the recorded auxiliary image is processed, if necessary, and transmitted to computer 14 and further to data printing and storage devices 15 and 16.
The first and second auxiliary fluorescent images are recorded in the same way as the fluorescent image of the tissue under examination (but with other recording objects).
The object position should not change when recording the fluorescent, colour reference, third and fourth auxiliary monochrome images.
The sequence of recording the fluorescent, colour reference, third and fourth auxiliary monochrome, first and second auxiliary fluorescent images can be different.
When looking for particular tissue segments (especially in case of endoscopic examination) the device operates in the view mode with continuous display of the colour and (or) fluorescent tissue image on the monitor screen.
The software of video, synchronisation and control signal processor 9 and computer 14 should support the operation of the claimed device with the use of the claimed method.
The aforementioned technical tasks are resolved by the proposed method due to the fact that, as compared with its analogues described above, firstly, in order to induce fluorescence, it uses long wave radiation in the wave length band of 630-645 nm which is within the fluorescence excitation band of only endogenous porphyrins and their complexes with proteins and does not induce interfering fluorescence of other endogenous fluorophores. However, it is known that it is the relative distribution of the concentration of endogenous porphyrins and their complexes with proteins in tissue that can provide information regarding the degree of proliferation of this or another tissue segment. Statistical processing of low level fluorescent signal allows to increase the real sensitivity of recording by averaging noise, since, according to classical statistics, the root-mean-square deviation of the number of independent events xcex94{overscore (n)} is in proportion to the square root of the number of events n, the relative value of fluctuations is in inverse proportionality to; {square root over (n)}:
xcex94n/nxcx9c1/{square root over (n)}
All this allows to record the fluorescent images reflecting the relative distribution of the natural concentrations of endogenous porphyrins and their complexes with proteins particularly in tissue and eliminates the need for preliminary preparation of the patient, as well as the need for invasive intervention into the patient""s organism, increases the precision and reliability of diagnostics. Besides, it eliminates the need for using expensive recording equipment with cooled receivers, brightness intensifiers etc. The diagnostic information can be easily documented and interpreted.
The use of the colour reference image of the tissue under examination combined with the fluorescent image allows to precisely locate proliferation areas and makes the method more convenient in practical use.
The use of the first auxiliary fluorescent image of the test object allows to monitor the process of diagnostics and achieve the consistency of recording results, monitor porphyrin exchange fluctuations in tissues and proliferal activity fluctuations in the patient""s organism over a long period of time.
The use of the second auxiliary fluorescent image allows to compare the degree of proliferation in the tissue under examination and the degree of proliferation in the healthy tissue of the same patient in the natural proliferation area. This allows to eliminate the effect of porphyrin exchange fluctuation factors on the results of diagnostics and the level of proliferative activity in the organism of a particular patient, that is, it allows to link the results of diagnostics to peculiarities in the organism of a particular patient.
The use of the third and fourth auxiliary images of the tissue under consideration allows to adjust the effect of local changes in optical absorption and scattering values of the tissue under examination in the spectral bands concerned on the fluorescent signal which increases the reliability of diagnostics.
The use of visible spectrum band without radiation in the wave length band of over 650 nm in the laboratory premises lighting source (FIG. 1) eliminates the need for working in complete darkness for the staff, since the laboratory premises should be fullly isolated from daylight (same as from other sources of interfering radiation in the wave length band which coincides with that in which fluorescent images are recorded).